Diagnostic and prognostic compounds and method

ABSTRACT

The present invention relates to a method of diagnosing bacterial or fungal sepsis in a subject, which method comprises the step of measuring the level of TREM-1-Ligand or TREM-1-Ligand nucleic acid in a biological sample obtained from said subject. The method can involve contacting said biological sample with a compound capable of binding TREM-1-Ligand and detecting the level of TREM-1-Ligand present in the sample by observing the level of binding between said compound and TREM-1-Ligand. Compounds, compositions and kits for use in the diagnosis of bacterial or fungal sepsis are also provided.

This invention relates generally to the field of immunology. Moreparticularly, the present invention relates to inflammation and the useof a specific marker as an indication of the activation state of myeloidcells. More specifically, the invention relates to markers that allowthe prompt diagnosis of sepsis of infectious (for example bacterial orfungal) origin and the follow up of septic patients duringpharmacological treatment.

Sepsis constitutes a significant consumption of intensive care resourcesand remains an ever-present problem in the intensive care unit. It hasbeen estimated that between 400 000 and 500 000 patients are so affectedeach year in both the USA and Europe. Morbidity and mortality haveremained high despite improvements in both supportive and anti-microbialtherapies. Mortality rates vary from 40% for uncomplicated sepsis to 80%in those suffering from septic shock and multi-organ dysfunction. Thepathogenesis of the conditions is now becoming better understood.Greater understanding of the complex network of immune, inflammatory andhaematological mediators may allow the development of rational and noveltherapies.

The condition of sepsis has previously been associated with many termsand nomenclature, reflecting both the complexity of the condition andthe similarity of the inflammatory response secondary to otheraetiologies. To illustrate the complex nature of sepsis, sepsis has beendefined by Edward O. Uthman, MD, as “a constellation of clinical andlaboratory findings from which an experienced physician concludes thatthe patient may have a serious infection”. His definition was purposelymade as a nebulous, subjective, and tautological definition, becauseattempts to define “sepsis” in the literature have stirred a great dealof disagreement and qualification.

In 1991, the American College of Chest Physicians and the AmericanSociety of Critical Care Medicine published definitions for systemicinflammatory response syndrome (SIRS) and sepsis, with the aim ofclarifying the diagnosis and treatment of these conditions and to aidinterpretation of research in this field (see Table 1).

A pattern of physiological variables have been shown in critically illpatients in response to a range of insults including; trauma, burns,pancreatitis and infection. These include inflammatory responses,leucocytosis or severe leucopaenia, hyperthermia or hypothermia,tachycardia and tachypnoea and have been collectively termed thesystemic inflammatory response syndrome (SIRS). This definitionemphasises the importance of the inflammatory process in theseconditions regardless of the presence of infection. The term sepsis isreserved for SIRS when infection is suspected or proven.

Sepsis is further stratified into severe sepsis when there is evidenceof organ hypoperfusion, made evident by signs of organ dysfunction suchas hypoxaemia, oliguria, lactic acidosis or altered cerebral function.Septic shock is severe sepsis complicated by hypotension defined assystolic blood pressure less than 90 mmHg despite adequate fluidresuscitation. Sepsis and SIRS may be complicated by the failure of twoor more organs, termed multiple organ failure (MOF), due to disorderedorgan perfusion and oxygenation. In addition to systemic effects ofinfection, a systemic inflammatory response may occur in severeinflammatory conditions such as pancreatitis and burns.

The appearance of signs of an inflammatory response is less well definedfollowing traumatic insults. In the intensive care unit, gram-negativebacteria are implicated in 50 to 60% of sepsis cases with gram-positivebacteria accounting for a further 35 to 40% of cases. The remainder ofcases are due to the less common causes of fungi, viruses and protozoa.

Early recognition of sepsis and Systemic Inflammatory Response Syndrome(SIRS) in the critically ill patient may avoid the increased morbidity,mortality and length of stay associated with multiple organ failure.However, there are major problems associated with diagnosis of sepsisand a clear need exists for rapid, reliable and sensitive methods todetect, monitor and treat SIRS due to infectious agents (sepsis).

TABLE 1 Definitions for the systemic inflammatory response syndrome(SIRS) and sepsis SIRS Two or more of: 1. Temperature > 38° C. or < 36°C. 2. Tachycardia > 90 beats/minute 3. Respiratory rate > 20breaths/minute or PaCO₂ < 4.3 kPa 4. White blood count > 12 × 10⁹/l or <4 × 10⁹/l or > 10% immature (band) forms Sepsis: SIRS due to infectionSevere sepsis: Sepsis with evidence of organ hypoperfusion Septic shock:Severe sepsis with hypotension (systolic BP < 90 mmHg) despite adequatefluid resuscitation or the requirement for vasopressors/inotropes tomaintain blood pressure

The present invention is directed towards circumventing the existingproblems associated with diagnosing sepsis to provide an accurate andconsistent method of detection.

The present invention is based upon the Inventors' surprising findingthat the ligand (referred to herein as “TREM-1-Ligand”) of a myeloidcell receptor called TREM-1 (referred to herein as “TREM-1-Receptor”) isa specific marker for bacterial and fungal sepsis (Systemic InflammatoryResponse Syndrome). In other words SIRS caused or exacerbated bybacterial or fungal infection.

Cells of the monocyte/macrophage lineage belong to the innate immunesystem and lack a highly diverse repertoire of antigen receptors.Nevertheless, their activity is regulated by a variety of activating andinhibitory cell surface receptors. Studies have identified a new familyof receptors, the TREM family, whose expression appears restricted tomyeloid cells (see Bouchon et al. (2000) J. Immunol. 164, 4991-4995).TREM receptors activate myeloid cells via association with the adaptormolecule DAP12.

Recent studies demonstrate that TREM-1-Receptor plays a critical role inthe inflammatory response to infection. Expression of TREM-1-Receptor isincreased on myeloid cells in response to both bacterial and fungalinfections in humans. Similarly, in mice the induction of shock bylipopolysaccharide (LPS) is associated with increased expression ofTREM-1-Receptor. Further, treatment of mice with a solubleTREM-1-Receptor/lmmunoglobulin fusion protein, as a ‘decoy’ receptor,protects mice from death due to LPS or E.coli. Significant survivalbenefit upon administration of the soluble receptor has been observed upto four hours after shock induction.

No ligands for TREM-1-Receptor have previously been identified.

As described herein the Inventors have detected TREM-1-Ligand onneutrophils isolated from peripheral neutrophils from patients withbacterial and fungal SIRS. The data described herein demonstrate that:the expression of TREM-1-Ligand has a diagnostic and prognostic value,since its expression is detected exclusively on circulating neutrophilsfrom patients with SIRS of bacterial or fungal origin (sepsis) and notin SIRS where no infection with extracellular pathogens (eg bacteria andfungi) could be proven. Therefore, its detection permits earlyrecognition of sepsis, allowing earlier intervention In addition, theexpression of TREM-1-Ligand on circulating neutrophils from patientswith sepsis is completely down-regulated when the patients show clinicalsigns of recovery. Therefore, the expression of TREM-1-Ligand alsoallows the monitoring and follow-up of septic patients during thepharmacological treatment of the disease.

Accordingly, the present invention provides methods and compositions forthe clinical screening and diagnosis of bacterial or fungal sepsis. Inaddition, the present invention provides methods and compositions formonitoring the effectiveness of bacterial or fungal sepsis treatment,for selecting participants in clinical trials relating to bacterial orfungal sepsis, for identifying subjects most likely to respond to aparticular therapeutic treatment for bacterial or fungal sepsis and forscreening and development of drugs for treatment of bacterial or fungalsepsis.

Thus, in a first aspect the invention provides a method of diagnosingbacterial or fungal sepsis in a subject, which method comprises the stepof measuring the level of TREM-1-Ligand or TREM-1-Ligand nucleic acid ina biological sample obtained from said subject.

In other words, the invention provides a method of diagnosing ormonitoring bacterial or fungal sepsis in a patient, comprising:measuring the level of TREM-1-Ligand or TREM-1-Ligand nucleic acid In asample from the patient, wherein the level is an indicator of presenceor extent of bacterial or fungal sepsis in the patient.

Furthermore, the invention provides a method of diagnosing bacterial orfungal sepsis in a subject which method comprises the step of measuringthe binding of a TREM-1 receptor-derived polypeptide to a sample ofcells, for example, neutrophils in a biological sample taken from apatient. Alternatively a measurement of the binding of a TREM-1receptor-derived polypeptide to the components, for example proteins, ofa cell-free sample obtained from a biological fluid, can be used todiagnose bacterial or fungal sepsis.

The methods of the invention can comprise the further step ofcorrelating said binding with the presence or absence of bacterial orfungal sepsis. This correlation can be made by comparing the measuredlevel in a biological sample taken from a patient with a mean level in acontrol sample or reference standard to indicate the presence or extentof bacterial or fungal sepsis in the patient.

The term “bacterial or fungal sepsis” as defined herein, means, SIRS(Systemic Inflammatory Response Syndrome) associated with infection byextracellular pathogens such as bacterial infection, for examplebacteremia (the presence of bacteria in the blood) with or without organfailure, and non-bacterial infections, such as fungemia (for example,yeast infection by Candida albicans), protozoal infections orparasitemia (such as in filariasis and trypanosomiasis) where increasedexpression of TREM-1-Ligand can be detected. Without wishing to be boundby theory, the Inventors suspect that TREM-1-Ligand expression is notusually increased in incidences of infection and sepsis caused byintracellular pathogens such as viruses.

“TREM-1-Ligand” as defined herein, is a ligand found on blood cells, inparticular circulating neutrophils, in patients with bacterial or fungalsepsis, which is bound by the TREM-1-Receptor. “TREM-1-Ligand nucleicacid” as defined herein is nucleic acid, i.e. mRNA or cDNA which ispresumed to be upregulated in patients with bacterial or fungal sepsisto bring about increased levels of “TREM-1-Ligand”.

In one embodiment, where the level of TREM-1-Ligand is measured, themeasurement of the level of TREM-1-Ligand comprises the steps of (a)contacting said biological sample with a compound capable of bindingTREM-1-Ligand; and (b) detecting the level of TREM-1-Ligand present inthe sample by observing the level of binding between said compound andTREM-1-Ligand.

The assay or measurement of the sample for the levels of TREM-1-Ligandpresent in the sample may be carried out using standard protocols knownin the art. For example, where the observation of binding betweenTREM-1-Ligand and the compound capable of binding TREM-1-Ligand takesplace, this observation may be carried out using known methodologies.For example the binding may be detected through use of a competitiveimmunoassay, a non-competitive assay system using techniques such aswestern blots, a radioimmunoassay, an ELISA (enzyme linked immunosorbentassay), a “sandwich” immunoassay, an immunoprecipitation assay, aprecipitin reaction, a gel diffusion precipitin reaction, animmunodiffusion assay, an agglutination assay, a complementfixationassay, an immunoradiometric assay, a fluorescent immunoassay, a proteinA immunoassay, an immunoprecipitation assay, an immunohistochemicalassay, a competition or sandwich ELISA, a radioimmunoassay, a Westernblot assay, an immunohistological assay, an immunocytochemical assay, adot blot assay, a fluorescence polarization assay, a scintillationproximity assay, a homogeneous time resolved fluorescence assay, a IAsysanalysis, and a BIAcore analysis.

For example, where an ELISA assay is performed, a TREM-1-Ligand bindingcompound is coated to a plate to capture TREM-1-Ligand from a biologicalsample. A different (e.g. labelled) or the same TREM-1-Ligand bindingcompound is then used to reveal the presence of TREM-1-Ligand on theplate, for example by it being conjugated to a detectable agent. It willbe understood by those skilled in the art that the TREM-1-Ligand bindingcompound need not be an antibody to be applicable in the immunoassaysmentioned above—a soluble form of the TREM-1 receptor, as describedherein, can be used in such assays.

In an alternative embodiment, where the level of TREM-1-Ligand nucleicacid is measured, the step of measuring the level of TREM-1-Ligandnucleic acid comprises the steps of (a) contacting said biologicalsample with an oligonucleotide probe or oligonucleotide primer specificfor TREM-1-Ligand nucleic acid and (b) detecting the level ofTREM-1-Ligand nucleic acid present in the sample by observing the levelof interaction between said oligonucleotide probe or oligonucleotideprimer and said TREM-1-Ligand nucleic acid. Such probes and primerspredominantly, preferably specifically, bind to TREM-1-Ligand nucleicacid in a manner sufficient to enable detection by known methods. The“level of interaction”, for example the level of binding of a probe orthe level of amplification brought about by a primer, provides anindication of the level or amount of nucleic acid (for example, cDNA orRNA) present in the sample and thus the level or amount of theTREM-1-Ligand. Such observations may be carried out using knownmethodologies and protocols. For example, where an oligonucleotide probeis used to detect the level of TREM-1-Ligand nucleic acid, for examplemRNA, Northern hybridizations, dot-blot, and in situ hybridizations canbe used. Where an oligonucleotide primer is used to detect TREM-1-Ligandnucleic acid, primer extension reactions, such as the polymerase chainreaction (PCR), for example quantitative PCR, can be carried out uponcDNA or RNA samples, to determine the level of TREM-1-Ligand nucleicacid.

The determination of the incidence of sepsis can be undertaken bycomparing the levels of TREM-1-Ligand or TREM-1-Ligand nucleic acidpresent in the sample with those in a control sample, the median levelin a group of control samples (for example, samples from healthyindividuals) or with data derived from previous analyses (for exampleprovided as a standard curve or illustration with a diagnostic kit ofthe invention or data within a computer program, for example associatedwith a diagnostic means of the invention). The determination of theincidence of sepsis may comprise deriving the likelihood ratio using amultivariate analysis based on distribution parameters from a set ofreference data derived from analysis of the levels of TREM-1-Ligand orTREM-1-Ligand nucleic acid in patients in which bacterial or fungalsepsis is absent, present or in remission.

The invention therefore also provides diagnostic means capable ofmeasuring levels of TREM-1-Ligand or TREM-1-Ligand nucleic acid and/orcomparing said levels to known levels that are indicative of the diseasestate of sepsis. Such diagnostic means can take the form of a sticktest, for example carrying the necessary reagents to perform the methodof the invention and to produce, for example, a colorimetric resultwhich can be compared against a colour chart. Other diagnostic meanswhich include a sample measuring means and/or a data processing meanscontaining standard data, as mentioned above, with associated programsfor comparing such data with data from a sample are also envisaged.

Thus, in either of the above embodiments, the method according to thefirst aspect of the invention can comprise the further step of c)correlating the detected level of TREM-1-Ligand or TREM-1-Ligand nucleicacid with the presence or absence of bacterial or fungal sepsis. Forexample, a correlation can be made by comparing the measured level ofTREM-1-Ligand or TREM-1-Ligand nucleic acid in the sample with a meanlevel in samples obtained from a control population of individuals nothaving bacterial or fungal sepsis, to indicate the presence or extent ofbacterial or fungal sepsis in the patient.

In a further embodiment, the method according to the first aspect of theinvention can be used in monitoring the progression or remission ofbacterial or fungal sepsis, in other words, to indicate the progressionor remission of the bacterial or fungal sepsis. Such methods can be usedto monitor the effectiveness and/or progress of bacterial or fungalsepsis therapy in a subject. In this embodiment, the method furthercomprises the steps of measuring the level of TREM-1-Ligand orTREM-1-Receptor nucleic acid in a second or further sample from thepatient, the first and second or further samples being obtained atdifferent times; and comparing the levels in the samples to indicate theprogression or remission of the bacterial or fungal sepsis.

The diagnostic methods according to the present invention are carriedout ex vivo. Biological samples for analysis by the methods of theinvention can be obtained using methods known in the art from varioussources, in particular from whole blood, blood serum, blood plasma,urine, cellular fractions of blood and neutrophils isolated fromperipheral blood. The sample should be a sample treated such that anyTREM-1-Ligand present is not removed prior to the assay or is renderedundetectable.

In addition to cell containing samples, the biological sample can be acell-free sample, for example a supernatant, obtained from, for example,a biological fluid. In this case, the components, for example proteins,of the sample are immobilised for example on a solid surface, forexample by coating onto a plastic surface. The presence of TREM-1-Ligandis then revealed using a TREM-1-Ligand binding compound, which isdetected via, for example an antibody or is itself conjugated to adetectable agent.

The methods of the invention are applicable to mammals, for examplehumans, non-human primates, sheep, pigs, cows, horses, goats, dogs, catsand rodents, such as mouse and rat. Generally, the biological sampletested by the methods of the invention is a human sample. In oneembodiment, the biological sample contains protein molecules from thetest subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or cDNA molecules from the test subject.

Preferably, the biological sample contains protein molecules from thetest subject. A preferred biological sample is a sample of granulocytesisolated from peripheral blood obtained by conventional means from asubject. A particularly preferred sample is a sample containingneutrophils.

In the present application, the term “compound capable of bindingTREM-1-Ligand” means polypeptides, ligands, antibodies or otherwisediscriminating entities which predominantly, preferably specifically,bind to TREM-1-Ligand. Such binding compounds, or “TREM-1-Ligand bindingpartners” can be a naturally occurring TREM-1-Ligand binding molecule,for example TREM-1-Receptor and natural and synthetic variants thereof.Further examples of binding compounds include, a chemically modified orgenetically modified derivative of a TREM-1-Ligand binding molecule, anartificially (for example chemically produced) TREM-1-Ligand bindingmolecule or a recombinant or engineered soluble TREM-1-Ligand bindingmolecule.

Included of use within the scope of the invention are antibodies whichbind predominately, preferably specifically or exclusively to,TREM-1-Ligand including, but not limited to, those antibodies which are:mono-or polyclonal antibodies (for example, raised againstTREM-1-Ligand), bi-specific, multi-specific, human, humanized, chimericantibodies, single chain antibodies, antibodies derived from phagedisplay techniques, Fab fragments, F(ab′)2 fragments, disulfide-linkedFvs, and fragments containing either a VL or VH domain or even acomplementary determining region (CDR) that specifically binds toTREM-1-Ligand. Such antibodies can be obtained according to methods wellknown in the art. (See, for instance, Chow, M. et al., Proc. Natl. Acad.Sci. USA 82:910-914; and Bittle, F. J. et al., J. Gen. Virol.66:2347-2354). Immunogenic epitopes of TREM-1-Ligand may be presentedtogether with a carrier protein, such as an albumin, to an animal system(such as rabbit or mouse) or, if long enough (at least about 25 aminoacids), without a carrier. However, immunogenic epitopes comprising asfew as 8 to 10 amino acids have been shown to be sufficient to raiseantibodies capable of binding to, at the very least, linear epitopes ina denatured polypeptide (e.g. in Western blotting.)

Otherwise modified immunoglobulins are also included within the scope ofthe invention, for example a fusion of the TREM-1-Receptor to one ormore immunoglobulin-derived protein domains, for example to confersolubility and/or stability, for example human IgG or IgM Fc fragments.

In addition, substances or products mimicking the tertiary structure ofthe TREM-1-Receptor can be used as binding partners specific forTREM-1-Ligand. It is possible to design such on the basis of computermodelling. The product can be produced synthetically using chemicalmeans. Use of recombinant DNA technology to engineer the requiredstructure is also possible as is chemical modification ofTREM-1-Receptor-like structures.

Furthermore, it is envisaged that isolated TREM-1-Ligand or computermodelling using the structure of TREM-1-Ligand, may be used to producebinding partners specific for TREM-1-Ligand using methods known in theart.

In a particular embodiment, a compound capable of binding TREM-1-Ligandcan be a modified variant of a naturally occurring TREM-1-Ligand bindingmolecule, for example the compound capable of binding TREM-1-Ligand canbe a polypeptide derived from the TREM-1-Receptor, referred to herein as“a TREM-1-Receptor-derived polypeptide”. Suitable“TREM-1-Receptor-derived polypeptides” are discussed further below.

The term “oligonucleotide probe or oligonucleotide primer specific forTREM-1-Ligand nucleic acid” includes any nucleic acid which is capableof binding specifically to TREM-1-Ligand nucleic acid in a mannersufficient to allow detection of TREM-1-Ligand nucleic acid in ahybridization reaction, a primer extension reaction or biochip-basedassay as known in the art.

Generally, the oligonucleotide probe or oligonucleotide primer specificfor TREM-1-Ligand nucleic acid will interact with TREM-1-Ligand nucleicacid in the sample under the stringent or moderately stringentconditions used in conventional DNA/RNA detection methods. With respectto hybridisation methods such as Northern or Southern analyses, the term“under stringent conditions” refers to hybridization and washingconditions under which nucleotide sequences having at least 60%,preferably 65%, more preferably 70%, most preferably 75% identity toeach other remain hybridized to each other. The term “moderatelystringent condition” refers to hybridization and washing conditionsunder which nucleotide sequences having at least 40%, preferably 45%,more preferably 50%, most preferably 55% identity to each other remainhybridized to each other. Such hybridization conditions are describedin, for example but not limited to, Current Protocols in MolecularBiology, 1989, John Wiley & Sons, New York, 6.3.1-6.3.6., and BasicMethods in Molecular Biology, 1986, Elsevier Science Publishing Co.,Inc., New York, 1986, pp. 75-78, and 84-87, and are well known to thoseskilled in the art. A preferred, non-limiting example of stringenthybridization conditions is hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C. followed by one or more washes in 0.2×SSC,0.1% SDS at about 50-65° C. A preferred, non-limiting example ofmoderately stringent conditions is hybridization in 6×SSC at about 42°C. followed by one or more washes in 0.2×SSC, 0.1% SDS at about 45-55°C.

With respect to primer extension reactions, stringent conditions willdepend on the primers used, but will be such that a TREM-1-Ligandnucleic acid template is preferentially amplified to allow detection.

According to a second aspect of the invention there is provided,compounds and pharmaceutical compositions for use in the diagnosis,prognosis, treatment or monitoring of the treatment of bacterial orfungal sepsis.

In one embodiment of this second aspect, the invention provides acompound capable of binding TREM-1-Ligand for use in the diagnosis,prognosis, treatment or monitoring of the treatment of bacterial orfungal sepsis. Also provided are oligonucleotide probes oroligonucleotide primers specific for TREM-1-Ligand for use in thediagnosis, prognosis, treatment or monitoring of the treatment ofbacterial or fungal sepsis.

In another embodiment, the invention provides use of a compound capableof binding TREM-1-Ligand or use of oligonucleotide probes oroligonucleotide primers specific for TREM-1-Ligand nucleic acid in amethod of diagnosis, prognosis, treatment or monitoring of bacterial orfungal sepsis.

In a further embodiment, the invention provides use of a compoundcapable of binding TREM-1-Ligand in the manufacture of a medicament forthe diagnosis, prognosis, treatment or monitoring of the treatment ofbacterial or fungal sepsis.

The methods described herein can furthermore be used as screening assaysto identify a subject with, or at risk of developing, bacterial orfungal sepsis. Such assays can be used to determine whether a subjectcan be administered an agent (e.g., an agonist, antagonist,peptidomimetic, protein, peptide, nucleic acid, small molecule, or otherdrug candidate) to treat bacterial or fungal sepsis. For example, suchmethods can be used to determine whether a subject can be effectivelytreated with a specific agent or class of agents (e.g., antibacterial orantifungal agents). Thus, the present invention provides methods fordetermining whether a subject can be effectively treated with an agentfor bacterial or fungal sepsis in which a test sample is obtained andthe TREM-1-Ligand or TREM-1-Ligand nucleic acid is detected

A further embodiment of the invention provides a composition (eg apharmaceutical composition) comprising a compound capable of bindingTREM-1-Ligand together with a pharmaceutically acceptable diluent,carrier or excipient for use in the diagnosis or treatment of bacterialor fungal sepsis.

Accordingly, also provided is the use of a compound capable of bindingTREM-1-Ligand in a method of treatment or diagnosis of bacterial orfungal sepsis. In other words, the use in diagnosis and treatment ofbacterial or fungal sepsis of a compound capable of bindingTREM-1-Ligand. The invention also provides a compound capable of bindingTREM-1-Ligand for use in, or used in, a method of diagnosis or treatmentof bacterial or fungal sepsis.

As used herein the language “pharmaceutically acceptable diluent,carrier or excipient” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions. Pharmaceutical compositions can be included in acontainer, pack, or dispenser together with instructions foradministration

A third aspect of the invention provides a method of identifyingmodulators of the expression and/or activity of TREM-1-Ligand saidmethod comprising comparing the level of binding in a sample containingsaid TREM-1-Ligand and a compound capable of binding TREM-1-Ligand, inthe presence and absence of a compound to be tested. Also provided byare agonists or antagonists of TREM-1-Ligand identified according to themethod of this aspect of the invention. Also provided is a method ofscreening compounds for use in bacterial or fungal sepsis therapycomprising determining the effect of those compounds on levels ofTREM-1-Ligand present in samples brought into contact with saidcompounds. Accordingly, the invention also provides a method of treatingbacterial or fungal sepsis in a subject, which method comprisesadministering to an individual in need thereof an effective amount of amodulator (eg an inhibitor) of expression or activity of TREM-1-Ligand.

Modulators of expression and/or activity include antagonists (eginhibitors) and agonists.

In a fourth aspect, the invention provides kits, associated reagents andcontacting means. In one embodiment the invention provides a kitcomprising at least one compound capable of binding TREM-1-Ligand andreagents for detecting binding of said compound to TREM-1-Ligand.

Another embodiment provides a kit comprising one or more oligonucleotideprobes or oligonucleotide primers specific for TREM-1-Ligand nucleicacid and reagents for detecting TREM-1-Ligand nucleic acid by means ofsaid probes or primers.

A further embodiment provides a kit comprising at least one compoundcapable of binding TREM-1-Ligand or one or more nucleotide probes orprimers specific for TREM-1-Ligand nucleic acid and means for contactingsaid compound or probes or primers with a sample containing said ligandor TREM-1-Ligand nucleic acid.

For TREM-1-Ligand binding compound-based kits, the kit can comprise, forexample: (1) a binding compound (e.g., attached to a solid support) thatbinds to TREM-1-Ligand; and, optionally, (2) a second, different bindingcompound e.g. an antibody, which binds to either the TREM-1-Ligand orthe first binding compound and is conjugated to a detectable agent.

For oligonucleotide-based kits, the kit can comprise, for example: (1)an oligonucleotide, e.g., a detectably labeled oligonucleotide, thathybridizes to a TREM-1-Ligand nucleic acid sequence; or (2) a pair ofprimers useful for amplifying a TREM-1-Ligand nucleic acid molecule. Thekit can also comprise, e.g., a buffering agent, a preservative, or aprotein stabilizing agent. The kit can also comprise componentsnecessary for detecting the detectable agent (e.g., an enzyme or asubstrate). The kit can also contain a control sample or a series ofcontrol samples which can be assayed and compared to the test samplecontained. Each component of the kit is usually enclosed within anindividual container, and all of the various containers are within asingle package, along with instructions for determining whether thesubject from which the sample is derived is suffering from or is at riskof developing bacterial or fungal sepsis.

As discussed above “TREM-1-Receptor-derived polypeptides” can functionas compounds capable of binding TREM-1-Ligand. In a presently preferredembodiment of the invention, a compound capable of binding TREM-1-Ligandis derived from the nucleic acid or amino acid sequence of humanTREM-1-Receptor (triggering receptor expressed on myeloid cells) forwhich the cDNA sequence is given in [SEQ ID NO:1]. The TREM-1-Receptoris expressed on human myeloid cells, is a transmembrane protein of theimmunoglobulin superfamily (Ig-SF). The TREM-1-Receptor is atransmembrane glycoprotein having the amino acid sequence of [SEQ IDNO:2] that is selectively expressed on blood neutrophils and a subset ofmonocytes but not on lymphocytes and other cell types.

Accordingly, the invention encompasses isolated or recombinantlyprepared TREM proteins or polypeptides or fragments, homologues,derivatives, or variants thereof, as defined herein, as“TREM-1-Receptor-derived polypeptides” Furthermore, this inventionencompasses nucleic acid molecules encoding the “TREM-1-Receptor-derivedpolypeptides” of the invention, and include cDNA, genomic DNA, and RNA.

In the description of TREM-1-Receptor-derived polypeptides that follows,in accordance with the definition of “compound capable of bindingTREM-1-Ligand”, the “TREM-1-Receptor-derived polypeptides” thus providedby the invention are those which predominantly, preferably specifically,bind TREM-1-Ligand.

Accordingly, TREM-1-Receptor-derived polypeptides can be encoded bynucleic acid molecules which comprise or consist of a nucleotidesequence that is about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 98% identical to the nucleotide sequence of [SEQID NO:1], or a complement thereof, or isolated nucleic acid moleculeswhich comprise or consist of about 25, 30, 35, 40, 45, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, or morecontiguous nucleotdes of the nucleotide sequence of [SEQ ID NO:1], or acomplement thereof.

TREM-1-Receptor-derived polypeptides can be encoded by a nucleotidesequence encoding a protein having an amino acid sequence that is atleast about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,or 98% identical to the amino acid sequence of [SEQ ID NO:2], orfragments, homologues, derivatives, or variants of said protein, orcomplement of said nucleic acid molecules.

TREM-1-Receptor-derived polypeptides can be encoded by nucleic acidmolecules comprising a nucleotide sequence encoding a protein having anamino acid sequence that comprises or consists of at least about 10, 15,20, 25, 30, 50, 75, 100, 125, 150, 175, 200, 225, 230 or more contiguousamino acids of [SEQ ID NO:2], or fragments, homologues, derivatives, orvariants of said protein, or complements of said nucleic acid molecules.

Furthermore, TREM-1-Receptor-derived polypeptides can be polypeptides orproteins comprising an amino acid sequence that is at least about 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identicalto the amino acid sequence of [SEQ ID NO:2], or fragments, homologues,derivatives, or variants thereof, or can be polypeptides or proteinscomprising an amino acid sequence that comprises or consists of at leastabout 10, 15, 20, 25, 30, 50, 75, 100, 125, 150, 175, 200, 225, 230 ormore contiguous amino acids of [SEQ ID NO:2], or fragments, homologues,derivatives, or variants thereof. In accordance with the statementabove, such fragments, homologues, derivatives or variants ofTREM-1-Receptor retain the ability or capability to bind TREM-1-Ligand.

The term “homologue,” especially “TREM-1-Receptor homologue” as usedherein refers to any member of a series of peptides or nucleic acidmolecules having the ability or capability to bind TREM-1-Ligand andhaving sufficient amino acid or nucleotide sequence identity as definedherein. TREM-1-Receptor homologues can be from either the same ordifferent species of animals.

The term “variant” as used herein refers either to a naturally occurringallelic variation of a given peptide or a recombinantly preparedvariation of a given peptide or protein in which one or more amino acidresidues have been modified by amino acid substitution, addition, ordeletion.

The term “derivative” as used herein refers to a variation of a givenpeptide or protein that is otherwise modified, i.e., by covalentattachment of any type of molecule, preferably having bioactivity, tothe peptide or protein, including non-naturally occurring amino acids.

The human TREM-1-Receptor cDNA is 884-nucleotide long (FIG. 11; [SEQ IDNO:1]) and the open reading frame of TREM-1-Receptor is nucleotides 48to 752 of [SEQ ID NO:1], which encodes a transmembrane proteincomprising the 234 amino acid sequence shown in FIG. 12 [SEQ ID NO:2].As shown in FIG. 12 [SEQ ID NO:2], the deduced amino acid sequence ofTREM-1 starts with a hydrophobic signal peptide at amino acid residues 1to 16 of [SEQ ID NO:2] ([SEQ ID NO:3]) followed by an extracellularregion composed of a single Ig-SF domain, encompassing amino acidresidues 17 to 200 of [SEQ ID NO:2] ([SEQ ID NO:4]), which contain threepotential N-glycosylation sites at amino acid residues 146 to 149 of[SEQ ID NO:2] (Asn-Ser-Thr-Gln; [SEQ ID NO:5]), 190 to 193 of [SEQ IDNO:2] (Asn-Leu-Thr-Asn; [SEQ ID NO:6]), and 193 to 196 of [SEQ ID NO:2](Asn-Val-Thr-Asp; [SEQ ID NO:7]), and the consensus sequences,Leu-Xaa-VaI-Xaa-Cys-Xaa-Tyr (at positions 37-43 of [SEQ ID NO:2]; “Xaa”indicates any amino acid) and Asp-Xaa-Gly-Xaa-Tyr-Xaa-Cys (at positions107-113 of [SEQ ID NO:2]), characteristic of the intrachain disulfidebridge of the Ig-SF V-type fold. The putative transmembrane domainstarts from amino acid residues 201 to 229 of [SEQ ID NO:2] ([SEQ IDNO:8]) and contains a charged lysine residue at position 217. Itscytoplasmic tail consists of 5 amino acid residues ([SEQ ID NO:9]) andappears to contain no signaling motifs.

A “signal sequence” or “signal peptide” as used herein refers to apeptide of at least about 10 to 40 amino acid residues which occurs atthe N-terminus of secretory or membrane-bound proteins and contains atleast about 50-75% hydrophobic amino acid residues such as alanine,leucine, isoleucine, phenylalanine, proline, tyrosine, tryptophan, orvaline. A signal sequence serves to direct a protein containing such asequence to a lipid bilayer. A signal sequence is usually cleaved duringthe maturation process of the protein. Thus, the invention also includesthe use of the domains and the mature protein resulting from cleavage ofsuch a signal peptide.

Accordingly, a mature TREM comprises one or more of the followingdomains: (1) an extracellular domain which contains at least one Ig-SFdomain; (2) a transmembrane domain; and (3) a cytoplasmic domain.

Thus, in one embodiment, a TREM-1-Receptor-derived polypeptide of use inthe invention comprises the amino acid sequence of [SEQ ID NO:2]. Inanother embodiment, a TREM-1-Receptor-derived polypeptide of theinvention is a mature polypeptide which does not contain a signalpeptide and comprises amino acid residues 17 to 234 of [SEQ ID NO:2]([SEQ ID NO:10]). In another aspect, a TREM-1-Receptor-derivedpolypeptide of the invention comprises the amino acid sequence of [SEQID NO:2] except that amino acid residues 1 to 16 of [SEQ ID NO:2] arereplaced by a heterologous signal peptide by genetic engineering.

In a particular and preferred embodiment, a TREM-1-Receptor-derivedpolypeptide of use in the invention comprises an extracellular domaincomprising amino acid residues 17 to 200 of [SEQ ID NO:2] ([SEQ IDNO:4]). In another embodiment, a TREM-1-Receptor-derived polypeptide ofthe invention comprises a transmembrane domain comprising amino acidresidues 201 to 229 of [SEQ ID NO:2] ([SEQ ID NO:8]).

Further, a TREM-1-Receptor-derived polypeptide of use in the inventioncomprises a cytoplasmic domain comprising amino acid residues 230 to 234of [SEQ ID NO:2] ([SEQ ID NO:9]).

In preferred embodiments, a TREM-1-Receptor-derived polypeptide of usein the invention comprises a fragment of [SEQ ID NO:2] which exhibitsbinding of TREM-1-Ligand. Such fragments are derived from theextracellular domain comprising amino acid residues 17 to 200 of [SEQ IDNO:2] ([SEQ ID NO:4]).

In addition to the TREM-1-Receptor-derived nucleic acid molecules andpolypeptides described above, other polypeptides or nucleic acidmolecules suitable for use in the invention are those polypeptides andnucleic acid molecules having the ability to bind (or express apolypeptide which binds) TREM-1-Ligand or TREM-1-Ligand nucleic acid.For example, these can be homologues of TREM-1-Receptor from either thesame or different species of animal, preferably from mammals, morepreferably from rodents, such as mouse [SEQ ID NO: 11] and rat, and mostpreferably from human.

Homologues of the TREM-1-Receptor nucleic acid molecule (i.e., [SEQ IDNO:1]) can be isolated based on their close nucleotide sequence identityto the human nucleic acid molecules disclosed herein, by standardhybridization techniques under stringent or moderately stringentconditions, as defined herein below, using the human cDNA or a portionthereof as a hybridization probe.

In another aspect, a variant of a TREM-1-Receptor-derived polypeptidecan be used in the methods of the invention in which the amino acidsequences have been modified by genetic engineering in order to eitherenhance or reduce biological activities of the polypeptides, or changethe local structures thereof whilst maintaining or retaining an abilityor capability to bind TREM-1-Ligand. Such modifications Include aminoacid substitution, deletion, and/or insertion. Amino acid modificationscan be made by any method known in the art and various methods areavailable to and routine for those skilled in the art.

For example, mutagenesis may be performed in accordance with any of thetechniques known in the art including, but not limited to, synthesizingan oligonucleotide having one or more modifications within the sequenceof a given polypeptide to be modified. Examples of mutagenesis used toobtain sequence variants include site specific mutagenesis, PCR-mediatedmutagenesis and treatment with mutagenic agents, such as hydroxylamine.

Preferably, the amino acid residues to be modified are surface exposedresidues. Additionally, in making amino acid substitutions, preferablythe amino acid residue to be substituted is a conservative amino acidsubstitution, for example, a polar residue is substituted with a polarresidue, a hydrophilic residue with a hydrophilic residue, hydrophobicresidue with a hydrophobic residue, a positively charged residue with apositively charged residue, or a negatively charged residue with anegatively charged residue. Moreover, preferably, the amino acid residueto be modified is not highly or completely conserved across speciesand/or is critical to maintain the biological activities of the protein.

Accordingly, suitable for use in the invention are nucleic acidmolecules encoding a TREM-1-Receptor-derived polypeptide that containsamino acid modifications that are not critical to activity. Thus, anisolated TREM-1-Receptor-derived nucleic acid molecule can be anucleotide sequence encoding a polypeptide having an amino acid sequencethat is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 98% identical to the amino acid sequence of [SEQ ID NO:2 or4] which has the ability to bind TREM-1-Ligand.

Furthermore, the invention also encompasses derivatives of theTREM-1-Receptor-derived polypeptides of the invention. For example, butnot by way of limitation, derivatives may include peptides or proteinsthat have been modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. Any of numerous chemical modifications maybe carried out by known techniques, including, but not limited to,specific chemical cleavage, acetylation, formylation, etc. Additionally,the derivative may contain one or more non-classical amino acids.

TREM-1-Receptor-derived polypeptides can be encoded by and expressedfrom recombinant expression vectors as known in the art which comprise anucleic acid encoding a TREM-1-Receptor-derived polypeptide in a formsuitable for expression of the nucleic acid in a host cell.

A variety of host-vector systems may be utilized in the presentinvention to express the protein-coding sequence. These include but arenot limited to bacteria transformed with bacteriophage, DNA, plasmidDNA, or cosmid DNA; microorganisms such as yeast containing yeastvectors; insect cell systems infected with virus (e.g., baculovirus); ormammalian cell systems infected with virus (e.g., vaccinia virus,adenovirus, etc.). The expression elements of vectors vary in theirstrengths and specificities. Depending on the host-vector systemutilized, any one of a number of suitable transcription and translationelements may be used. Alternatively, the recombinant expression vectorcan be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

In an particular embodiment of the invention, TREM-1-Receptor-derivedpolypeptides can be a fusion protein comprising a bioactive molecule andone or more domains of TREM-1-Receptor or fragment thereof. Inparticular, the present invention provides fusion proteins comprising abioactive molecule (for example, conferring stability, solubility oracting as a reporter moiety or an additional binding moiety)recombinantly fused or chemically conjugated (including both covalentand non-covalent conjugations) to one or more domains of TREM-1-Receptoror fragments thereof.

Thus, the present invention further encompasses fusion proteins in whichthe TREM-1-Receptor-derived polypeptides of the invention or fragmentsthereof, are recombinantly fused or chemically conjugated (includingboth covalent and non-covalent conjugations) to heterologouspolypeptides (i.e., an unrelated polypeptide or portion thereof,preferably at least 10, at least 20, at least 30, at least 40, at least50, at least 60, at least 70, at least 80, at least 90 or at least 100amino acids of the polypeptide) to generate fusion proteins. The fusiondoes not necessarily need to be direct, but may occur through linkersequences.

In one example, a fusion protein in which a TREM-1-Receptor-derivedpolypeptide of the invention or a fragment thereof can be fused tosequences derived from various types of immunoglobulins. For example, aTREM-1-Receptor-derived polypeptide of the invention can be fused to aconstant region (e.g., hinge, CH2, and CH3 domains) of IgG1 or IgMmolecule (human or murine), for example, as described in Examples 1, and2 herein, so as to make the fused polypeptides or fragments thereof moresoluble and stable in vitro and in vivo.

A particular TREM-1-Receptor-derived polypeptide is described in Example3, in which a fusion protein between the extracellular portion ofTREM-1-Receptor and the constant domain of human IgG1(TREM-1-Receptor-huIgG1) is used to detect levels of TREM-1-Ligand.

In one aspect, the fusion protein comprises a TREM-1-Receptor-derivedpolypeptide of the invention which is fused to a heterologous signalsequence at its N-terminus. For example, the signal sequence naturallyfound in the polypeptide of the invention can be replaced by a signalsequence which is derived from a heterologous origin. Various signalsequences are commercially available. For example, the secretorysequences of melittin and human placental alkaline phosphatase(Stratagene; La Jolla, Calif.) are available as eukaryotic heterologoussignal sequences. As examples of prokaryotic heterologous signalsequences, the phoA secretory signal (Sambrook, et al., supra; andCurrent Protocols in Molecular Biology, 1992, Ausubel, et al., eds.,John Wiley & Sons) and the protein A secretory signal (PharmaciaBiotech; Piscataway, N.J.) can be listed. Another example is the gp67secretory sequence of the baculovirus envelope protein (CurrentProtocols in Molecular Biology, 1992, Ausubel, et al., eds., John Wiley& Sons).

In another embodiment, a TREM-1-Receptor-derived polypeptide of theinvention can be fused to tag sequences, e.g., a hexa-histidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), or the hemagglutinin “HA” tag, whichcorresponds to an epitope derived from the influenza hemagglutininprotein (Wilson, et al., 1984, Cell 37:767) and the “flag” tag (Knappik,et al, 1994, Biotechniques 17(4):754-761). These tags among others, manyof which are commercially available, are especially useful forpurification of recombinantly produced TREM-1-Receptor-derivedpolypeptides.

Fusion proteins can be produced by standard recombinant DNA techniquesor by protein synthetic techniques, e.g., by use of a peptidesynthesizer. Once a fusion protein of the invention has been produced byrecombinant expression, it may be purified by any method known in theart for purification of a protein, for example, by chromatography (e.g.,ion exchange, affinity, particularly by affinity for the specificantibody, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins.

Aspects of the invention can be also applied in the framework ofmultiple diagnosis of a subject. For example, in a method of screening apatient for presence or susceptibility to disease, comprising performinga plurality of diagnostic tests on a tissue sample from the patient fora plurality of diseases, the invention provides the improvement whereinone of the diagnostic tests comprises measuring the level ofTREM-1-Ligand or TREM-1-Ligand nucleic acid.

The various aspects and embodiments of the invention described abovealso apply to the following: a diagnostic means for detecting bacterialor fungal sepsis; a diagnostic kit comprising such a diagnostic means; amethod of treatment of infection, which includes the step of screeningan individual for bacterial or fungal sepsis, wherein sepsis iscorrelated with the levels of TREM-1-Ligand or TREM-1-Ligand nucleicacid in a sample from said individual, and if sepsis is identified,treating that individual to prevent or reduce the infection; and theuse, in the manufacture of means for detecting sepsis, of a compoundcapable of binding TREM-1-Ligand.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

Preferred features of each aspect of the invention are applicable toeach other aspect, mutatis mutandis.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described with reference to thefollowing non-limiting examples, with reference to the figures, inwhich:

FIG. 1. shows a scheme of the cecal ligation and puncture (CLP) mousemodel of sepsis. C57BLU6 mice were anaesthetised and the caecum wasexposed through an abdominal midline incision and subjected to a 50-80%ligation of the distal half followed by a single puncture with a G23needle. A small amount of stool was expelled from the punctures toensure patency. Then the caecum was replaced into the peritoneal cavityand the abdominal incision closed in layers. Survival after CLP wasassessed 4-6 times a day for at least 7 days.

FIG. 2. shows a visualization of murine TREM-1-Ligand in normal andseptic mice. Panel A) Whole blood staining. Cells in whole blood weredouble stained with murine TREM-1-Receptor/IgG fluorescent tetramer andLy-6G (top plots) or human IgG fluorescent tetramer and Ly-6G (bottomplots). The frequencies of the double positive population in onerepresentative control mouse and one septic (CLP) mouse are indicated.Panel B) Peritoneal cell staining. Cells in the peritoneal cavity ofeither normal or septic (CLP) mice were stained with murineTREM-1-Receptor/IgG or human TREM-1-Receptor/IgM. The overall frequencyof Ly-6G positive, murine TREM-1-Receptor/IgG positive cells in arepresentative control mouse is 8.8% compared to 35% that were detectedin peritoneal cells derived from a representative septic (CLP) mouse.Histogram plots show the frequency of double positive populations in thelow Ly-6G (40% in the control mouse and 77% in the septic mouse) andhigh Ly-6G (33% in the septic mouse) expressing cell subsets.

FIG. 3. shows a time course of the expression of TREM-1-Ligand onperitoneal granulocytes of septic mice. Cells were recovered fromperitoneal lavage and whole blood of septic (CLP) mice at differenttimes (30 min, 3 hrs, 6 hrs) after CLP induction and then stained withLy-6G-PE and mTREM-1-Receptor/IgG-tetramer-Alexa 488. Panel A showshistogram plots showing the percentages of mTREM-1-Receptor-IgG-tetramerpositive cells within the gated Ly-6G positive cell population in septicmice. Panel B shows a time course of TREM-1-Ligand expression inperitoneal lavage and whole blood of septic mice.

FIG. 4. shows that TREM-1-Ligand is expressed on peripheral neutrophilsfrom patients with sepsis: dose-dependent staining with solublehuTREM-1/IgM. Neutrophils isolated from peripheral blood of patientswith sepsis were stained with different concentrations of huTREM-1/IgM:0.2, 0.5, 1 μg/ml. Histogram plots represent expression of TREM-1-Ligandin one representative patient with sepsis in the acute phase and at thetime of recovery.

FIG. 5. shows expression of TREM-1-Ligand on peripheral neutrophils ofpatients with sepsis. Histograms plots represent expression ofTREM-1-Ligand in the 12 individual patients with sepsis included in thestudy. Neutrophils isolated from peripheral blood as described herein,were stained with huTREM-1/IgM and analyzed by flow cytometry (emptyhistograms) at the time of acute disease and after recovery. The secondtime point is missing in two patients whose blood samples were not madeavailable because the patients died of septic shock. Filled histogramsrepresent staining with control IgM.

FIG. 6. shows expression of TREM-1-Ligand in a representative patientwith Systemic Inflammatory Response Syndrome (SIRS) not associated withsepsis (central hyperthermia). Peripheral blood neutrophils wereanalyzed as described in the legend to FIG. 5.

FIG. 7. shows TREM-1-Ligand expression on peripheral blood neutrophilsof patients with sepsis and SIRS of non-infectious origin. Individualdata of TREM-1-Ligand expression are reported. Data are reported asratio between the geometric mean fluorescence of cells stained withhuTREM-1-Receptor/IgM and the geometric mean fluorescence of cellsstained with control IgM. A statistically significant difference wasobserved between the sepsis in the acute phase and sepsis at recovery(p<0.0001) and between sepsis and SIRS (p<0.0001). The sepsis group alsodiffers significantly from the healthy control group (p<0.001).Statistical analysis was performed with Kruskal-Wallis test.

FIG. 8. shows a time course of TREM-1-Ligand expression on peripheralblood neutrophils of patients with sepsis. The Geometric MeanFluorescence (GMF) of TREM-1-Ligand in sepsis patients in the acutephase and at the time of recovery is reported. Down-regulation ofTREM-1-Ligand is observed in all septic patients analyzed, except inthose two cases where a second sample was not made available because thepatients died.

FIG. 9. shows TREM-1-Receptor and TREM-1-Ligand are expressed bydifferent subsets of neutrophils in human sepsis and in the CLP mouse.Panel A) Expression of TREM-1-Ligand was evaluated on peripheralneutrophils of septic patients (peripheral pmn, sepsis patients) bydouble staining with anti human CD15 MAb and huTREM-1/IgM. Dot plotsindicate that two different subsets of CD15 positive cells can bedistinguished in septic patients: low CD15 and high CD15 expressingcells. The histogram plots indicate the percentages of TREM-1-Ligandexpressing cells in the two subsets. The majority of TREM-1-Ligandexpressing cells are found in the CD15 low subset (38%), while barely nocells are present in CD15 high subset. Panel B) Expression ofTREM-1-Ligand was evaluated on peripheral neutrophils of CLP mice (CLPperitoneal lavage) by double staining with anti mouse Ly-6G andmTREM-1-Receptor/IgG. Dot plots indicate that two different subsets ofLy-6G positive cells can be distinguished in CLP mice: low Ly-6G andhigh Ly-6G expressing cells. The histogram plots indicate thepercentages of TREM-1-Ligand expressing cells in the two subsets. Themajority of TREM-1-Ligand expressing cells are found in the Ly-6G lowsubset (33%), while barely no cells are present in the Ly-6G highsubset.

FIG. 10. shows in vitro induction of TREM-1-Ligand expression. In vitrostimulation of whole blood from healthy donor with total crude extractsof Gram-positive and Gram-negative bacteria from blood cultures ofseptic patients. Whole blood from a representative healthy donor wasstimulated as described herein for 16 hours, cells were stained withhuTREM-1-Receptor/IgM and analyzed by flow cytometry. Cells were gatedbased on physical parameters (SSC, side scatter and FSC, forwardscatter). Histogram plots in panel A represent analysis of granulocytes(high SSC). Histogram plots in panel B represent analysis of lymphocytes(low SSC).

FIG. 11. shows Human TREM-1-Receptor cDNA [SEQ ID NO:1].

FIG. 12. FIGS. 12A and 12B show Human TREM-1-Receptor amino acidsequences [SEQ ID NO: 2].

FIG. 13. shows Human TREM-1-Receptor (signal peptide) amino acidsequence [SEQ ID NO:3].

FIG. 14. shows Human TREM-1-Receptor (extra-cellular region) amino acidsequence [SEQ ID NO:4].

FIG. 15. shows Human TREM-1-Receptor (N-glycosylation site) amino acidsequence [SEQ ID NO:5], Human TREM-1-Receptor (N-glycosylation site)amino acid sequences [SEQ ID NO:6, 7 and 8], and Human TREM-1-Receptor(cytoplasmic tail) amino acid sequence [SEQ ID NO:9].

FIG. 16. FIGS. 16A and 16B show Human TREM-1 Receptor (mature protein)amino acid sequence [SEQ ID NO: 10].

FIG. 17. shows Murine TREM-1-Receptor cDNA sequence [SEQ ID NO:11](Genbank Accession No. NM_(—)021406)

FIG. 18. shows immunofluorescence stainings performed with aTREM-1-tetramer construct. Briefly, Fc tail or mTrem1/IgG (mouseTREM-1/IgG Fc constant region fusion protein) are incubated with ProteinA FITC at a molar ratio of 4:1 for 30 min at RT. The reactions areblocked with Human IgG for 15 min at RT. The complexes are transferredonto the samples and incubated for 1 hr on ice. Granulocytes are washedand counter-stained with anti GR-1 PE.

FIG. 19. shows the preparation of cDNA for subtraction. mRNA is isolatedusing Microfast-Track™ (Invitrogen). cDNA is synthesized and amplifiedusing SMART™ PCR Synthesis kit (BD Bioscience Clontech) following themanufacturer's instructions. Amplified cDNA is then prepared forsubtraction using both the SMART™ PCR cDNA Synthesis kit and thePCR-Select™ cDNA Subtraction kit following the manufacturer'sinstruction. Briefly, cDNA is digested with RsaI and purified. Differentadaptors are ligated to the ends of separated populations of targetcDNA. Lane 1=Mw markers; lane 2=tester, lane 3=subtracted; lane4=driver.

FIG. 20. shows real time PCR of mouse beta-actin in both subtracted andun-subtracted populations.

FIGS. 21 (TREM-1-Ligand positive) and 22 (TREM-1-Ligand negative). showthe screening of a library performed using ³²P probes from un-subtractedor subtracted cDNA as described in Example 4. Solid squares=genesexpressed in CLP mice but not in normal mice; dotted squares=genesexpressed in normal mice but not in CLP mice.

EXAMPLES Example 1 TREM-1-Ligand is Expressed on Neutrophils of SepticMice

Methods

Production of murine TREM-1-Receptor/human IgG fusion protein To producemurine TREM-1-Receptor (mTREM-1-Receptor) [SEQ ID NO:11] as a solublefusion protein, a chimeric gene consisting of the mTREM-1-Receptorextracellular domain (GenBank accession number NM_(—)021406) and humanIgG constant regions was constructed using the plasmid pCD4 (Trauneckeret al., 1991, Trends Biotechnol., 9:109) which is derived from plasmidpHT4-Y1 which can be prepared as previously described by Traunecker,Lüke and Karjalainen in Nature 331, 84-86 (1988) and EP0394827. The cDNAfragment encoding the mTREM-1-Receptor extracellular region wasamplified by PCR from cloned plasmid DNA. The forward primer(TAGTAGAAGCTTATACTTACCGTCAGCATCTGTCCCATTTAT) [SEQ ID NO: 12] contained aHindIII restriction site and the TREM-1 start codon. The reverse primer(TAGTAGGAATTCAGGATGAGGMGGCTGGG) [SEQ ID NO: 13] provided an EcoR1restriction site. The ˜640-bp PCR product was cut with HindIII andEcoRI, and ligated into an expression vector containing the exons forhinge, CH2 and CH3 regions of human IgG, the guanosinephosphotransferase gene conferring resistance to mycophenolic acid, andthe k promoter for the expression in the mouse myeloma cell line J558L.Transfection, screening of culture supernatants and purification ofmTREM-1-Receptor-IgG were performed as described (Bouchon et al., Nature410: 1103, 2001).

Quantification of mTREM-1-Receptor/IgG Fusion Protein.

Purified murine IgG fusion proteins were assayed for specificity, titerand functionality by ELISA using anti human IgG as a capturing proteinand specific biotinylated mAb against mTREM-1-Receptor (50D1, ratIgG1,κ) followed by streptavidin-HRP. Immunoblot analysis of purifiedhuman IgG fusion proteins revealed only one band of immunoreactivity.

Cecal Ligation and Puncture (CLP).

CLP was performed as described previously (FIG. 1). Briefly, C57BU6 micewere anaesthetised by intraperitoneal administration of 75 mg/kgKetanest® (Parke Davies & Company, Munich, Germany) and 16 mg/kg Rompun®(Bayer A G, Leverkusen, Germany) in 0.2 ml sterile pyrogen-free saline(B. Braun Melsungen AG, Melsungen, Germany). The caecum was exposedthrough a 1.0-1.5 cm abdominal midline incision and subjected to a50-80% ligation of the distal half followed by a single puncture with aG23 needle. A small amount of stool was expelled from the punctures toensure patency. Then the caecum was replaced into the peritoneal cavityand the abdominal incision closed in layers with 5/0 Prolene thread(Ethicon, Norderstedt, Germany). 500 μl sterile saline containing 1 mgof mTREM-1-Receptor-IgG1 or 1 mg huIgG1,κ (Sigma) was injectedintraperitoneally immediately after CLP. The CLP was performed blindedto the identity of the treatment group. Survival after CLP was assessed4-6 times a day for at least 7 days.

Analysis of Blood and Peritoneal Lavage Fluids

Blood (250 μl) was collected from the tail vein of mice into a SerumSeparator Tube (Becton Dickinson) at different time points afterinduction of CLP. Peritoneal lavage cells were harvested at differenttime points after CLP induction. Total cell numbers were determined on aCoulter counter and differential counts were performed according tostandard morphological criteria on cytospin preparations stained withGiemsa & May-Gruenwald solution (Sigma). A minimum of 200 cells werecounted per field, with 3 fields per sample for peritoneal lavage.

Visualization of Murine TREM-1-Ligand in Normal and Septic Mice.

For whole blood staining, 150 μl of 1:2-diluted blood from both normalmice (right plots) and septic mice (left plots) were incubated withFcγIII/II block Mab for 20 min RT. Blood cells were further stained with8 μg/sample of mTREM-1-Receptor/IgG (top plots) and huIgG (bottom plots)fluorescent tetramers. Briefly, 32 μg of murine TREM-1-Receptor-IgG orhuman IgG as a control, were complexed with Protein A Alexa488-conjugated (Molecular Probes) at 4:1 molar ratio for 30 min at RT inPBS/BSA 0.5% buffer, in the dark. Multimer staining were carried out in150 μl PBS/BSA buffer for 1 hr on ice, in the presence of further 2 μghuIgG per sample, in order to completely block non specific bindingsites of protein A. Samples were washed twice with PBS/BSA and furtherstained with anti Ly-6G-PE antibody. Red blood cells were eventuallylysed by using BD lysing solution, before analysing the samples bycytofluorimetric analysis. Cells recovered from peritoneal cavity ofeither normal or CLP-treated mice were blocked with anti-FcγIII/II MAb(10 μg/ml) (Pharmingen) for 20 min at room temperature. Cells werefurther stained with mTREM-1-Receptor/IgG or huTREM-1-Receptor/IgMsupernatants (fusion protein concentration >40 μg/ml), 1 ml/sample, 20min on ice. After wash, samples were incubated with anti human IgG-PEand anti Ly-6G-FITC antibody under standard staining conditions, andthen subjected to cytofluorimetric analysis.

Results

The ligand for murine TREM-1 is up-regulated under septic conditions, inthe cecal ligation and puncture model (CLP) (FIG. 1). By using twodifferent staining approaches, a mTREM-1-Receptor/IgG tetramer (panel A)and monomer (panel B), we detected cells that specifically bind themTREM-1-Receptor fusion protein, indicating the presence of ligand onthe cell surface (FIG. 2). Staining is particularly evident inneutrophils obtained from the peritoneal cavity of septic mice. FewTREM-1-Ligand positive appear to be present also in normal, non-septicconditions, but at a much lower extent compared to septic mice. HumanTREM-1 fusion protein does not stain murine cells and it is used as anegative control under the same staining conditions. As shown in FIG. 3,TREM-1-Ligand expression appears to be upregulated in peritoneal cellsfrom septic mice between 3 and 6 hours after CLP induction. The kineticsof TREM-1-Ligand expression in cells isolated from the blood of septicmice is slower since 6 hours after CLP induction, only 2% of Ly-6Gpositive cells express TREM-1-Ligand. However, TREM-1-Ligand is welldetected in cells isolated from the blood of septic mice 15 hours afterCLP induction.

Example 2 TREM-1-Ligand is Expressed on Circulating Neutrophils ofPatients with Septic Shock

Methods

Production of Human TREM-1-Receptor/Human IgM Fusion Protein

To produce human TREM-1-Receptor (huTREM-1) as a soluble fusion protein,the cDNA fragment encoding the huTREM-1 (GenBank accession number AF196329) extracellular region [SEQ ID NO: 6] was amplified by PCR withforward primer (TAGTAGGAGCTCACAGGAAGGATGAGGAAGACCAGGCTC) [SEQ ID NO: 14]containing an SstI restriction site and blunt reverse primer(AAGCTTATACTTACCCCTGATGATATCTGTCACATTTGT) [SEQ ID NO: 15] and clonedinto an expression vector containing the exons for hinge, CH2, and CH3region of human IgM. This vector is a derivative of plasmid pCD4(Traunecker et al., 1991, Trends Biotechnol., 9:109) which is derivedfrom plasmid pHT4-Y1 which can be prepared as previously described byTraunecker, Loke and Karjalainen in Nature 331, 84-86 (1988) andEP0394827. Transfection of the chimeric gene into the mouse myeloma cellline J558L, screening of culture supernatants, and purification ofhuTREM-1/IgM were performed as previously described (Traunecker et al.,1991, Trends Biotechnol., 9:109).

Quantification of Human TREM-1/Human IgM Fusion Proteins

Purified human IgM fusion proteins were assayed for specificity, titerand functionality by ELISA using anti-human IgG/IgM (JacksonLaboratories) as a capturing protein and a specific biotinylated mAbagainst huTREM-1-Receptor (21C7, murine IgG1,κ), followed bystreptavidin-HRP. Immunoblot analysis of purified human IgM fusionproteins revealed only one band of immunoreactivity.

Isolation of Peripheral Blood Neutrophils.

Peripheral blood from 26 patients with Systemic Inflammatory ResponseSyndrome (SIRS) were analyzed: 12 patients with associated systemicbacterial/fungal infections (sepsis) and 14 with SIRS from differentclinical insults without any evidence of systemic bacterial and fungalinfection. Peripheral blood was collected soon after diagnosis of SIRSand sepsis, before initiation of any antibiotic and steroid treatment.All patients had body temperature>38° C., heart rate >90/min, and whitecell count >12×10⁹/I. A second peripheral blood sample was obtainedafter recovery, defined as normalization of the above clinicalparameters. Expression of TREM-1-Ligand was evaluated at the abovedescribed time-points.

Staining of Peripheral Blood Neutrophils.

Peripheral granulocytes were isolated from blood by dextran sulfate andsubsequent Ficoll gradient. Purified cells were then stained with antiTREM-1 Mab and huTREM-1/IgM. Briefly, cells were pre-incubated withhuman IgG (Sigma) to block free Fc binding sites. Supernatant (100 μl=1μg) of soluble TREM-1/IgM was added and incubated 30 minutes at 4° C.After washing, 1 μl of F(ab)₂ donkey anti-human IgG1-PE (JacksonImmunoresearch) were added. After washing, cells were resuspended andanalyzed by flow cytometry (FACS LSR, Becton Dickinson). Negativecontrol was performed staining granulocytes with purified human IgM. Insome cases, double staining with anti CD15 (Pharmingen) was performed.

Statistical Analysis

Statistical analysis was performed by Kruskal-Wallis test.

Results

Peripheral blood neutrophils obtained from 14 patients with SIRS(Systemic Inflammatory Response Syndrome) and 12 patients with sepsiswere analyzed for expression of TREM-1 and TREM-1-Ligand. Patients wereclassified based on the criteria indicated in FIG. 4. TREM-1 isconstitutively expressed on peripheral neutrophils and its expression isonly minimally regulated during SIRS or sepsis. Peripheral neutrophilsfrom patients with SIRS and sepsis were incubated with solublehuTREM-1/IgM fusion protein in order to detect surface expression ofTREM-1-Ligand. The staining with huTREM-1/IgM was specific and dosedependent. As shown in FIG. 4, the number of positive cells wasproportional to the amount of huTREM-1/IgM used for the staining. With 1μg huTREM-1/IgM, 80% of the cells stained positive, while with 0.2 μg,only 45% of the cells stained positive. A high percentage of peripheralneutrophils isolated from patients with sepsis stained positive withhuman TREM-1/IgM, indicating that they expressed TREM-1-Ligand on thecell surface (FIG. 5). However, when peripheral neutrophils from thesame patients were analyzed at the time of recovery, the percentage ofthe cells expressing TREM-1-Ligand was dramatically reduced. Thispattern of TREM-1-Ligand expression was observed in all of the 12patients with sepsis that were analyzed in the study, independently ofthe bacterial strain that was isolated from their blood culture (Table2).

TABLE 2 Bacterial strains isolated from blood culture of patients withsepsis. A total of 12 patients with sepsis were analyzed in the study.All of them had an identifiable bacterial strain isolated from theirblood cultures. In one case Candida albicans was isolated from the bloodculture. PATIENT ETHIOLOGY 1 Pseudomonas aeruginosa 2 Staphilococcusaureus 3 Pseudomonas aeruginosa 4 Candida albicans 5 Serratia marcescens6 Staphilococcus aureus 7 Staphilococcus aureus 8 Escherichia coli 9Neisseria meningitidis 10 Haemophilus influenzae 11 Streptococcuspyogenes 12 Staphilococcus aureus

These data indicate that down-regulation of the expression ofTREM-1-Ligand associated with positive outcome of the disease.Interestingly, TREM-1-Ligand expression was not detected on peripheralneutrophils from patients with SIRS resulting from a range of insults,including trauma, meningitis, pneumonia (Table 3 and FIG. 6). Therefore,TREM-1-Ligand expression was a specific marker of sepsis.

TABLE 3 Clinical diagnosis of patients with Systemic InflammatoryResponse Syndrome (SIRS) not associated with sepsis. A total of 14patients with SIRS were analyzed in the study. The patients wereadmitted to the Intensive Care Unit with different diagnoses that arelisted here. No bacteria/fungi were isolated from their blood. DIAGNOSISNUMBER OF PATIENTS Neurological coma 2 Central hypertermia 3 Meningitis1 Multiple Organ Failure 2 Trauma 2 Pneumonia 2 Not diagnosed 2

The expression of TREM-1-Ligand in patients with sepsis in the acutephase and after recovery, and in patients with SIRS is summarized inFIG. 7, where individual data of expression are reported. Data arereported as ratio between the geometric mean fluorescence of cellsstained with huTREM-1/IgM and geometric mean fluorescence of cellsstained with control IgM. A statistically significant difference wasobserved between the sepsis in the acute phase and sepsis at recovery(p<0.0001) and between sepsis and SIRS (p<0.0001). The sepsis group alsodiffers significantly from the healthy control group (p<0.001).

A time course of the expression of TREM-1-Ligand in the sepsis patientsis reported in FIG. 8. It is clearly evident that expression ofTREM-1-Ligand drops in all sepsis patients at the time of recovery. Thedecrease of TREM-1-Ligand expression is observed in all septic patientsanalyzed, except in those two cases where a second sample was not madeavailable because the patients died.

Notably, the expression of TREM-1-Ligand seems to be restricted to asubset of neutrophils during sepsis. As shown in FIG. 9, panel A, 38% ofCD15 low neutrophils isolated from a septic patient expressedTREM-1-Ligand, while as few as 2% of CD15 high neutrophils expressedTREM-1-Ligand. The same pattern of TREM-1-Ligand expression is alsodetected on neutrophils from CLP mice (FIG. 10; panel B), where themajority of TREM-1-Ligand positive cells belong to the Ly-6G low subsetof cells isolated from peritoneal lavage (77%), while only 33% of theLy-6G high cells express TREM-1-Ligand.

In conclusion, the data presented here demonstrate that the expressionof TREM-1-Ligand has a diagnostic value, since its expression isdetected exclusively on circulating neutrophils from patients with SIRSof bacterial origin (sepsis) and not on neutrophils from patients withSIRS where bacterial infection could not be proven. Therefore, detectionof TREM-1-Ligand expression permits early recognition of sepsis, animportant issue in the management of the condition allowing earlierintervention.

In addition, the expression of TREM-1-Ligand has a prognostic value,because its expression on circulating neutrophils from patients withsepsis is completely down-regulated when the patients show clinicalsigns of recovery. Consequently, the monitoring of the expression ofTREM-1-Ligand allows the follow-up of septic patients during thepharmacological treatment of the disease as well as assessment ofcurrent and novel therapies.

Example 3 Induction of TREM-1-Ligand Expression in vitro

Methods

In vitro Stimulation of Whole Blood with Crude Bacterial Extracts.

Bacteria isolated from blood cultures or bronchial exudates of septicpatients were collected and crude extracts were prepared by repeatingfreezing and thawing. Total crude extracts were used for in vitrostimulation of whole blood. Briefly, whole blood was diluted 1:6 incomplete RPMI medium. Crude bacterial extracts (1 μg/ml) were addeddirectly to whole blood and incubated for 16 hours at 37/40° C. Stainingwith soluble TREM-1/IgM was performed as described above. Samples wereanalyzed by flow cytometry (FACS LSR, Becton Dickinson).

Results

In order to analyze the time course of the expression of TREM-1-Ligand,we set up an in vitro system in which TREM-1-Ligand expression can beinduced by defined stimuli. Whole blood from healthy donors wasstimulated overnight with E.coli LPS or total crude bacterial extractsfrom Gram-positive bacteria, Gram-negative bacteria and fungi derivedfrom blood cultures or bronchial exudates of septic patients.TREM-1-Ligand expression was evaluated by cytofluorimetric analysis ongranulocytes and lymphocytes from peripheral blood. As shown in FIGS. 10(panel A), expression of TREM-1-Ligand was induced on granulocytes byGram⁺, Gram⁻ bacteria and fungi. TREM-1-Ligand expression isspecifically induced on granulocytes, since its expression was notdetected on peripheral lymphocytes stimulated under the sameexperimental conditions (FIG. 10, panel B) Addition of proinflammatorycytokines (TNFα, IL-1β, IL6, IL-12) during the stimulation onlypartially increased TREM-1-Ligand expression on granulocytes (data notshown). TREM-1-Ligand expression was detected as early as 2 hours afterstimulation of whole blood with bacterial extracts and was detectable upto 16 hours after initial stimulation (data not shown). These dataindicate that TREM-1-Ligand expression is promptly unregulated on thecell surface on neutrophils upon encounter with bacterial antigens invitro.

Example 4 TREM-1 Ligand Identification: Generation of a DifferentialExpression Library of CLP Versus Normal Neutrophils

TREM-1 ligand expression on murine neutrophils is tightly regulated:TREM-1 ligand is not present on peripheral neutrophils of normal mice orbone marrow derived mature granulocytes (FIG. 18).

TREM-1 ligand can be detected on neutrophils of the peritoneal cavity onCLP treated mice, with a peak of expression 5 hours after CLP induction.TREM-1 ligand can be identified among those genes that aredifferentially expressed between these two populations and followed byscreening the gene for TREM-1 binding.

Generation of Subtracted cDNA Libraries.

Subtracted hybridization and suppressive PCR are performed using thePCR-select cDNA subtraction kit according to the manufacturersinstructions (Clontech). This procedure combines subtractivehybridization and suppressive PCR in order to enrich for differentiallyexpressed genes. Briefly, cDNA from the CLP versus normal mice arefragmented with Rsal, ligated with adaptors and then are subjected toalternate rounds of hybridization and PCR amplification. The subtractionprotocol allows only molecules in the test population that did nothybridize with molecules in the control population to be exponentiallyamplified in subsequent cycles of PCR, resulting in a dramatic loss ofcommon background sequences. As demonstrated by electrophoresis gelanalysis (FIG. 19) the banding pattern of the subtracted populationresults in distinct bands rather than the smear produced by nonsubtracted cDNA. Furthermore, subtraction efficiency is confirmed by PCRto compare the abundance of the housekeeping gene β-actin in cDNAsamples before and after subtraction (FIG. 20). The subtractedpopulation is then cloned into a plasmid vector to generate a plasmidiclibrary and plated in a 96 well plate format.

Differential Screening of Subtracted cDNA Clones.

Randomly selected clones, for example 400, are then screened to detectthose differentially expressed in the TREM-1 Ligand positive population.Screening of the library is performed using ³²P probes fromun-subtracted or subtracted cDNA (FIG. 21).

Densitometry analysis allows the selection of clones that can besequenced and compared to known sequences in public databases. Thecombined protocol of subtractive hybridization, suppressive PCR anddifferential screening is effective in removing commonly expressedgenes. Many genes can be represented more than once among the subtractedclones. However, because of the nature of the subtraction procedure, thefrequency with which clones matched a particular database entry is notan accurate reflection of expression levels for genes within the targetpopulation.

The differential gene expression observed can be validated usingreal-time PCR. Candidate gene(s) are then screened for TREM-1 binding byperforming both in vitro transcription/translation and cytofluorimetricanalysis on transfected cells as known in the art.

1. A method of diagnosing bacterial or fungal sepsis in a subject, whichmethod comprises the step of measuring the level of TREM-1-Ligand in abiological sample obtained from said subject.
 2. The method of claim 1wherein said step of measuring the level of TREM-1-Ligand comprises thesteps of: (a) contacting said biological sample with a compound capableof binding TREM-1-Ligand; (b) detecting the level of TREM-1-Ligandpresent in the sample by observing the level of binding between saidcompound and TREM-1-Ligand.
 3. The method of claim 2, comprising thefurther step of: c) correlating the detected level of TREM-1-Ligand withthe presence or absence of bacterial or fungal sepsis.
 4. The method ofclaim 3 where said correlation is made by comparing the measured levelof TREM-1-Ligand in the sample with a mean level in a control sample orreference standard to indicate the presence or extent of bacterial orfungal sepsis in the patient.
 5. The method of claim 2 wherein saidcompound specifically binds TREM-1-Ligand.
 6. The method of claim 2wherein said compound capable of binding TREM-1-Ligand is selected fromthe group consisting of: (a) a TREM-1 ligand binding fragment of SEQ IDNO: 2 or a homologue thereof; (b) a fusion protein comprising abioactive molecule and one or more TREM-1 ligand binding domains of SEQID NO: 2 or a homologue thereof or a TREM-1 ligand binding fragmentthereof; (c) a fusion protein comprising a detectable label and one ormore TREM-1 ligand binding domains of SEQ ID NO: 2 or a homologuethereof or a TREM-1 ligand binding fragment thereof.
 7. The method ofclaim 2 wherein said compound capable of binding TREM-1-Ligand is aTREM-1-Receptor/immunoglobulin fusion protein.
 8. The method of claim 7wherein said fusion protein comprises the sequence of FIG. 14 [SEQ IDNO: 4].
 9. The method of claim 2 wherein said method is selected fromthe group consisting of a competitive immunoassay, western blots, aradioimmunoassay, an ELISA (enzyme linked immunosorbent assay), a“sandwich” immunoassay, an immunoprecipitation assay, a precipitinreaction, a gel diffusion precipitin reaction, an immunodiffusion assay,an agglutination assay, complement fixation assay, an immunoradiometricassay, a fluorescent immunoassay, a protein A immunoassay, animmunoprecipitation assay, an immunohistochemical assay, a competitionor sandwich ELISA, a radioimmunoassay, a Western blot assay, animmunohistological assay, an immunocytochemical assay, a dot blot assay,a fluorescence polarization assay, a scintillation proximity assay, ahomogeneous time resolved fluorescence assay, a IAsys analysis, and aBIAcore analysis.
 10. The method of claim 1, further comprising thesteps of measuring the level of TREM-1-Ligand in a second or furthersample from the patient, the first and second or further samples beingobtained at different times; and comparing the levels in the samples toindicate the progression or remission of the bacterial or fungal sepsis.11. The method of claim 1 wherein said method is a method of diagnosingbacterial sepsis in a subject.
 12. The method of claim 1 wherein thesample is selected from the group consisting of whole blood, bloodserum, blood plasma, urine, cellular fractions of blood and neutrophils.13. The method of claim 1 wherein the sample is a cell-free sampleobtained from a biological fluid.
 14. The method of claim 1 wherein thesample is a human sample.
 15. A method of diagnosing bacterial or fungalsepsis in a subject which method comprises the step of measuring thebinding of a TREM-1 receptor-derived polypeptide to a sample ofneutrophils in a biological sample taken from a patient.
 16. The methodof claim 15, comprising the further step of correlating said bindingwith the presence or absence of bacterial or fungal sepsis.
 17. Themethod of claim 16 where said correlation is made by comparing themeasured level in a biological sample taken from a patient with a meanlevel in a control sample or reference standard to indicate the presenceor extent of bacterial or fungal sepsis in the patient.